330
Related Techniques
- Primer pairs (set 1 (mammalian aldolase primer): 5'-CCG GAG CAG
AAG AAG GAG CT-3' and 5'-CAC ATA CTG GCA GCG CTT CA-3';
set 2 (mammalian prolactin receptor primer): 5'-CTG GGA CAG AT
G GAG GAC-3' and 5'-CTC AGG TTT TAA TCG AAT TT-3'; set 3 (m
ammalian IL-l~ primer): 5'-ATG AGG ATG ACT TGT TCT TT-3' an
d 5'-GAG GTG CTG ATG TAC CAG TT-3')
Miscellaneous - DNA isolation kit (Gentra Systems)
Preparation
of cells
DNA
extraction
- GeneAmp PCR reagent kit (Perkin Elmer)
- Metaphor gel (FMC Bioproducts)
- Type 55 positive/negative film (Polaroid Corporation)
• • Procedure
1. Culture cells to confluency.
2. Harvest the cells by suspending them in the culture medium, and by
centrifuging them (150 g 5min), and wash them with physiological
saline.
1. Prepare between 3 x 10 6 and 5 x 10 6 cells in the case of relatively large
cells such as most lepidopteran cells, and between 12 x 10 6 and 15 x 10 6
cells for small cells, such as some dipteran cell lines.
2. Extract DNA from the cells using a non-phenol extraction protocol of
a DNA isolation kit. The absorbance ratio (at 260/280) ranges from 15
to l.9 for DNA samples.
Fig. 2. DNA amplification fingerprinting profiles of
various insect cell lines with the primers set as follows: (aldolase) 5' -CCGGAGCAGAAGAAGGAGCT3' and 5' -CACATACTGGCAGCGCTTCA-3'. Water,
negative control without genome DNA; marker,
DNA marker; D.m Line 2, Schneider's Drosophila
melanogaster cell line; A. aegypti 59, Peleg and
Shahar's Aedes aegypti cell line; A. albopictus ATC15, Singh's Aedes albopictus cell line; BRL-AG-l,
Stiles et al:s Authonomus grandis cell line; DSIRHA-1179, Crawford's Heteronychus arator cell line;
AC-20, Chiu and Black's Agal/ia constricta cell line.
From Mcintosh, A.H. et al. (1996) "Identification of
insect cell lines by DNA amplification fingerprinting (DAF)", Insect Molecular Biology, Vol.5, 187-195,
copyright 1996 by Blackwell Science Ltd. Reproduced with permission of copyright owner
Related Techniques
- Primer pairs (set 1 (mammalian aldolase primer): 5'-CCG GAG CAG
AAG AAG GAG CT-3' and 5'-CAC ATA CTG GCA GCG CTT CA-3';
set 2 (mammalian prolactin receptor primer): 5'-CTG GGA CAG AT
G GAG GAC-3' and 5'-CTC AGG TTT TAA TCG AAT TT-3'; set 3 (m
ammalian IL-l~ primer): 5'-ATG AGG ATG ACT TGT TCT TT-3' an
d 5'-GAG GTG CTG ATG TAC CAG TT-3')
Miscellaneous - DNA isolation kit (Gentra Systems)
Preparation
of cells
DNA
extraction
- GeneAmp PCR reagent kit (Perkin Elmer)
- Metaphor gel (FMC Bioproducts)
- Type 55 positive/negative film (Polaroid Corporation)
• • Procedure
1. Culture cells to confluency.
2. Harvest the cells by suspending them in the culture medium, and by
centrifuging them (150 g 5min), and wash them with physiological
saline.
1. Prepare between 3 x 10 6 and 5 x 10 6 cells in the case of relatively large
cells such as most lepidopteran cells, and between 12 x 10 6 and 15 x 10 6
cells for small cells, such as some dipteran cell lines.
2. Extract DNA from the cells using a non-phenol extraction protocol of
a DNA isolation kit. The absorbance ratio (at 260/280) ranges from 15
to l.9 for DNA samples.
Fig. 2. DNA amplification fingerprinting profiles of
various insect cell lines with the primers set as follows: (aldolase) 5' -CCGGAGCAGAAGAAGGAGCT3' and 5' -CACATACTGGCAGCGCTTCA-3'. Water,
negative control without genome DNA; marker,
DNA marker; D.m Line 2, Schneider's Drosophila
melanogaster cell line; A. aegypti 59, Peleg and
Shahar's Aedes aegypti cell line; A. albopictus ATC15, Singh's Aedes albopictus cell line; BRL-AG-l,
Stiles et al:s Authonomus grandis cell line; DSIRHA-1179, Crawford's Heteronychus arator cell line;
AC-20, Chiu and Black's Agal/ia constricta cell line.
From Mcintosh, A.H. et al. (1996) "Identification of
insect cell lines by DNA amplification fingerprinting (DAF)", Insect Molecular Biology, Vol.5, 187-195,
copyright 1996 by Blackwell Science Ltd. Reproduced with permission of copyright owner